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Yeast Alcohol Dehydrogenase Structure and Catalysis

Savarimuthu Baskar Raj,† S. Ramaswamy,‡ and Bryce V. Plapp*
Department of Biochemistry, The University of Iowa, Iowa City, Iowa 52242, United States
*
S Supporting Information

ABSTRACT: Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (ADH1)

is the constitutive enzyme that reduces acetaldehyde to ethanol during the
fermentation of glucose. ADH1 is a homotetramer of subunits with 347 amino
acid residues. A structure for ADH1 was determined by X-ray crystallography at
2.4 Å resolution. The asymmetric unit contains four different subunits, arranged
as similar dimers named AB and CD. The unit cell contains two different
tetramers made up of “back-to-back” dimers, AB:AB and CD:CD. The A and C
subunits in each dimer are structurally similar, with a closed conformation,
bound coenzyme, and the oxygen of 2,2,2-trifluoroethanol ligated to the catalytic
zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66.
In contrast, the B and D subunits have an open conformation with no bound
coenzyme, and the catalytic zinc has an alternative, inverted coordination with
Cys-43, Cys-153, His-66, and the carboxylate of Glu-67. The asymmetry in the
dimeric subunits of the tetramer provides two structures that appear to be
relevant for the catalytic mechanism. The alternative coordination of the zinc may represent an intermediate in the mechanism of
displacement of the zinc-bound water with alcohol or aldehyde substrates. Substitution of Glu-67 with Gln-67 decreases the
catalytic efficiency by 100-fold. Previous studies of structural modeling, evolutionary relationships, substrate specificity, chemical
modification, and site-directed mutagenesis are interpreted more fully with the three-dimensional structure.

NAD(P)-dependent oxidoreductases occur in virtually all structure could not be determined by molecular replacement
organisms and catalyze the reversible oxidation of primary (despite the availability of several ADH structures) or by
and secondary alcohols into aldehydes and ketones, respec- multiple isomorphous replacement. We found one, only
tively. Medium-chain alcohol dehydrogenases (ADHs, EC partially twinned, crystal with different cell dimensions and
1.1.1.1) contain 327−376 amino acid residues per chain and determined the structure by molecular replacement using the
are usually zinc-dependent.1 The ADHs in higher eukaryotes structure of the tetrameric Pseudomonas aeruginosa ADH.16 The
(plants and animals) are usually dimeric, whereas those in structure of yeast ADH1 now permits comparisons with other
prokaryotes and lower eukaryotes (yeast) are tetrameric. The ADHs and a better understanding of previous studies of ADH1.
dimeric horse liver ADH (374 amino acid residues, 80000 Da)
was the first of this superfamily to be studied by X-ray
crystallography and is a model for the other ADHs.2−4 In yeast,
■ EXPERIMENTAL PROCEDURES
Protein Preparation. The gene for Saccharomyces cerevisiae
constitutive ADH1 catalyzes the reduction of acetaldehyde to ADH1 (adc1, YOL086c; UniProtKB entry P00330), the
ethanol during the fermentation of glucose. The enzyme was constitutive, fermentative enzyme from the laboratory strain
purified and crystallized with ammonium sulfate, and its kinetic, of baker’s yeast, was expressed from plasmid YEp13 in host
chemical, and physical properties have been studied exten- yeast that did not express any of the three medium-chain, zinc-
sively.5−8 Yeast ADH1 is a tetramer of four identical subunits containing alcohol dehydrogenases,8,10,17 and the protein was
with 347 amino acid residues each and a calculated mass of purified to homogeneity as described previously.18
147396 Da. The amino acid and gene sequences of yeast ADH1 Crystallization. Crystals were obtained by the hanging
were determined.9,10 Molecular modeling shows that yeast drop, vapor diffusion method using Fluka polyethylene glycol
ADH is homologous to horse liver ADH,11 but deletion of 21 5000 monomethyl ether (MPEG-5000) as the precipitating
amino acid residues from the catalytic domain of yeast ADH1 agent. The hanging drop contained 16 μL of 10 mg/mL protein
and other gaps and insertions make exact comparisons in 125 mM sodium N-tris(hydroxymethyl)methyl-3-amino-
problematic. propanesulfonic acid buffer (pH 8.4) (at 25 °C), 1.7 mM
Yeast ADH1 was one of the first enzymes to be crystallized.5 nicotinamide 8-iodoadenine dinucleotide (8ID, kindly provided
Thin, hexagonal crystals were also found in anaerobically grown by N. J. Oppenheimer), 0.1 M 2,2,2-trifluoroethanol (TFE),
yeast, apparently with a tetrahedral arrangement of subunits in
the P312 space group.12,13 Crystals that diffracted well for X-ray Received: May 27, 2014
crystallography were reported previously,14,15 and we collected Revised: August 22, 2014
many data sets; however, most crystals were twinned, and a Published: August 26, 2014

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0.16 mM EDTA, and 7% MPEG-5000 and was equilibrated NAD, but with 0.5 M TFE in an attempt to stabilize the ternary
over 0.73−0.84 mL of a reservoir solution containing 22−26% complex.
MPEG-5000 and 0.1 M TFE. Crystals grew in 2 weeks at 4 °C. The asymmetric unit contains four crystallographically
Crystals were soaked in ∼1 mL of the same buffer with 30% different, but structurally similar, subunits arranged as two
MPEG-5000 and 0.5 M TFE (but no 8ID because no more was dimers that we named AB and CD and are similar to the
available) for 5 days during their shipment on ice from Iowa to dimeric horse liver ADH (Figure 1A). Each subunit in a dimer
Germany before flash vitrification in liquid N2 and data has a coenzyme binding domain typical of the Rossmann fold
collection. (six-stranded parallel β-pleated sheet with two helices on each
Data Collection and Structure Determination. X-ray side of the sheet) to which the coenzyme binds at the carboxyl
data were collected July 28, 1995, on synchrotron beamline terminal end.23 Extensive interactions of the two coenzyme
BW7A at the EMBL/DESY Hamburg unit using a single crystal binding domains produce an extended β-sheet in a dimer. Each
at 100 K and a 300 mm MAR Research imaging plate detector subunit also has a catalytic domain that contains the zinc atom
and a wavelength of 0.8570 Å. The data were processed with to which the alcohol binds and a structural zinc in a distant
d*TREK.115 The structure was determined using molecular loop. The substrates bind in the cleft between the domains. The
replacement using AMORE19 and P. aeruginosa ADH [Protein A and C subunits have a “closed” conformation and contain
Data Bank (PDB) entry 1LLU, sequence 42% identical to that NAD and TFE bound to the catalytic zinc in the “classic”
of yeast ADH1] as the search model. The estimated solvent coordination, whereas subunits B and D have an “open”
content of the crystals is 53%, and the Matthews coefficient, conformation with TFE in the substrate binding pocket near
VM, is 2.62 Å3/Da. O20 and REFMAC21 were used for model the catalytic zinc, which has an alternative coordination, and no
building and refinement, respectively. Refinement was bound coenzyme. Packing considerations lead to the conclusion
improved by including the twin operator (0.713 for h, k, l that the unit cell contains three biological molecules each of
and 0.287 for −h, −k, l) and by using eight TLS elements two different tetramers with AB:AB and CD:CD subunits
representing the catalytic domains, residues 1−154 and 294− (Figure 1B).
347, and the coenzyme binding domain, residues 155−293, for Subunit and Dimer Structures. Superpositioning of the
each of the four subunits. See Table 1 for data collection and α-carbon atoms of the coenzyme or catalytic domains, or the
refinement statistics. complete subunits, shows that the conformations of the A and
C subunits (containing NAD and TFE) are very similar to one
Table 1. X-ray Data Collection and Refinement Statistics another (average rmsd of 0.30 Å); the B and D subunits
(containing only TFE) are also similar to one another [rmsd of
PDB entry 4W6Z
0.54 Å (see Table 1S of the Supporting Information)]. The
space group P321
higher rmsd value for B and D subunits relative to that for the A
no. of different subunits in the asymmetric unit 4
and C subunits results from disorder, or poor electron density,
cell dimensions (Å) 144.39, 144.39, 128.20
for residues in some loops of the D subunit: 49−59, 246−251,
cell angles (deg) 90, 90, 120
and 269−274.
resolution range (Å) 28.1−2.4
The dimers have similar structures, as superposition of
no. of measured reflections (total, unique) 229310, 59568
dimers AB and CD gives an rmsd value of 0.45 Å. However, the
completeness (%) (outer shell) 98.5 (92.7)
A and C subunits differ somewhat from the B and D subunits in
Rmeas (outer shell)a 0.134 (0.484)
coenzyme and catalytic domain structures (average rmsd of
mean ⟨I⟩/σ⟨I⟩ (outer shell) 8.0 (3.5)
0.87 Å) and in the overall subunits (average rmsd of 1.97 Å).
Rvalue, Rfree, test (%)b 0.178, 0.222, 2.5
rmsdc for bond distances (Å), angles (deg) 0.015, 1.82
The conformation of the A and C subunits is more closed than
no. of protein atoms 10328
that of the B and D subunits. After the coenzyme binding
8 Zn, 2 NAD(8-Iodo), 4 TFE atoms 122
domains of subunits A and B are superimposed, a rotation of
no. of waters 151
∼13° around an effective hinge axis24 superimposes the
mean B value (Å2), Wilson, REFMAC 39.4, 40.2
catalytic domains of the A and B subunits. Horse liver ADH
estimated coordinate error (Å) 0.15
was the first enzyme shown in atomic detail by X-ray
a crystallography to undergo a conformational change when
Rmeas = Rrim, with redundancy-independent merging.115 bRvalue = (∑|
substrates bind; a rotation of ∼10° closes up the substrate
F0 − kFc|)/∑|F0|, where k is a scale factor. Rfree was calculated with the
indicated percentage of reflections not used in the refinement.116 binding cleft.3 The extent of conformational change for ADHs
c
Root-mean-square deviations from ideal geometry. from different sources varies; the rotation of the open and
closed subunits differs by ∼7° in cod liver ADH and ∼12° in
Escherichia coli ADH.25−27 The conformational change is

■ RESULTS AND DISCUSSION

Structure Solution Overview. The diffracting crystals of
important for coenzyme binding and catalytic efficiency.28
Quaternary Interactions. The packing of the symmetry-
related dimers in the unit cell could produce two different
ADH1 reported previously14 in space group P622 turned out to tetramers: a “back-to-back” dimer of dimers, AB:AB and
be twinned,22 and the structure could not be determined. CD:CD, in which the active sites are on the “front” sides and
Extensive attempts to prepare other crystal forms and to fully available to bind substrates; or “front-to-front” dimers,
prepare heavy atom derivatives were also not successful except AB:CD, in which the active sites would be opposed and less
for one crystal that was prepared in the presence of accessible to solvent. The sensible choice favors the back-to-
trifluoroethanol and nicotinamide 8-iodoadenine dinucleotide back arrangement (Figure 1B), and this fits with calculations of
(8ID in the PDB file, hereafter termed NAD), which we buried surface area (Table 2S of the Supporting Information).
thought might be useful for phasing. This crystal was unusual in The formation of the dimers (AB or CD) buries ∼3400 Å2 of
that it was shipped in crystallization media that did not contain the 30000 Å2 of combined area of the monomers. The
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Biochemistry Article

Figure 1. Stereoviews of one asymmetric unit, an AB dimer, and of the biologic AB:AB tetramer in a back-to-back orientation. (A) In the AB dimer,
the A subunit has bound NAD and TFE (in ball and stick representation) and a closed conformation, whereas the B subunit has only TFE and an
open conformation. The zinc atoms are shown as gray spheres. (B) In the tetramer, the catalytic subunits of the two A subunits (blue, with NAD and
TFE colored green) are most closely associated with one another in the tetramer, and the catalytic domains of B subunits (magenta) are likewise
associated. These figures were made with the PyMOL Molecular Graphics System, version 1.7, from Schrödinger, LLC.

formation of the AB:AB or CD:CD tetramers buries ∼4400 Å2 extended coenzyme binding domain. Such interactions were
of the 54000 Å2 of combined area of the dimers. On the other also identified in horse liver ADH in the βS and βF elements in
hand, the formation of an AB:CD tetramer would bury only horse liver ADH, linked by a short 310-helix.2
∼1700 Å2 of the combined area. Each tetramer contains two Side chains of several residues (101−110) from the structural
different subunits, A1B1:A2B2 or C1D1:C2D2 (1 and 2 being zinc binding loop of the catalytic domain of one subunit also
symmetry-related subunits), in a quasi-tetrahedral arrangement, interact with residues (260−262) of the coenzyme binding
about a noncrystallographic, molecular 2-fold axis (near domain of the other subunit in a dimer. The carboxylate of Glu-
residues 277 and 290). The back-to-back arrangement of 101 forms a salt bridge with the guanidinium group of Arg-260
dimers in the tetramer is also found in the other tetrameric between subunits of both dimers. The side chain of Asn-110 in
ADHs. subunit A (or C) interacts with side chains of Asn-262 and Ser-
The buried surface area is an indicator of the fit between 287 in subunit B (or D). Because the conformations of the
protein subunits, but the specific interactions between subunits subunits are slightly different, due to the relative rotations of
in the dimers and tetramers are important for understanding the catalytic and coenzyme binding domains within a subunit,
oligomer formation and the heat stability of yeast ADH1 in the interactions between subunits are not symmetrical. Some
comparison to those of the more stable yeast ADH2. Most of water-mediated hydrogen bonds link Gly-237 and Ala-261
the interactions between subunits A and B (or C and D) in the backbone atoms to Tyr-102. Altogether, the intradimer
dimers involve residues from the coenzyme binding domains of interactions amount to 21−22 hydrogen bonds, 2−4 electro-
the Rossmann fold (Figure 1S of the Supporting Information). static interactions, 14−15 “hydrophobic” interactions (van der
Several hydrogen bonds connect the peptide backbones of the Waals interactions, ≤∼4 Å), and one disulfide bond.
antiparallel β-strands that include residues 274−280 and 288− The disulfide bond is formed between Cys-277 residues
292 around the dimeric molecular 2-fold axis. A short α-helix across the molecular 2-fold axis in a dimer (Figure 2S of the
links these two β-strands. These interactions produce the Supporting Information). As presented in the Supporting
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Biochemistry Article

Information, a disulfide bond was also found in the purified and Nevertheless, the differences in the horse and yeast enzymes
crystallized commercial enzyme. Treatment of ADH1 with 10 are reflected in some shifts in helices and sheets so that the
mM dithiothreitol at pH 7.0 decreased the t50 (temperature at subunits do not exactly superimpose.
which 50% of enzyme activity was lost) from ∼59 to ∼38 °C None of the substitutions in yeast ADH appears to generate
and promoted dissociation to inactive subunits, making the interactions that would explain why yeast ADH is a tetramer
stability of enzyme similar to that of the chimeric ADH1(1− whereas horse ADH is a dimer. The deletion of 21 amino acid
258)−ADH2(259−347) that has Cys-277 substituted with residues from the horse enzyme, which leads to differences in
Ser.29 However, this chimeric enzyme also has six other residues 114−122 in yeast ADH, was suggested to be
substitutions, which may destabilize the enzyme. [Yeast ADH1 responsible for the tetrameric structure of yeast ADH.33
and ADH2 differ in 24 amino acid residues, and ADH2 has a t50 Multiple-sequence alignments of alcohol dehydrogenases were
of ∼70 °C (see below also).] The disulfide bond may not be consistent with this possibility, indicating a distant divergence
important for heat stability, as the cysteine residue is not in evolution of dimers and tetramers.32,34 A recent analysis has
conserved in medium-chain ADHs, including thermophilic labeled this particular sequence as the “quaternary structure-
ADHs. determining loop”,35 but the structural basis needs to be
As illustrated in Figure 1B, the tetramer has the symmetry- established. Superpositioning the horse liver dimer onto the
related AB:AB dimers with subunit A1 most closely juxtaposed corresponding dimers in the yeast tetramer shows that the extra
with subunit A2, and B1 with B2, but there are also interactions loop would probably not prevent formation of a tetrameric
of A1 with B2 and A2 with B1. The interactions between horse enzyme, as the subunits could fit together, with a few
dimers that produce the tetramers are also extensive (Figure 1S good interactions and some close contacts that could be
of the Supporting Information). Interactions within the CD:CD accommodated by amino acid substitutions and small structural
tetramer are similar, and we identify approximately 4−6 changes. Because the oligomeric state and the length of the
electrostatic interactions, 16−20 hydrogen bonding interac- residues in the loop region are not strictly correlated,35,36 it
tions, and 30−34 hydrophobic interactions in each tetramer. would be interesting to explore the interconversion of dimeric
The tetrameric enzyme dissociates to enzymatically inactive and tetrameric ADHs by protein engineering and to create
dimers or monomers upon mild heat treatment, and yeast active yeast ADH dimers and liver ADH tetramers. We do not
ADH1 is less stable than ADH2.29 Construction of seven yet understand the evolutionary basis or significance for the
different chimeric ADH1/2 enzymes showed that the heat formation of the oligomeric enzymes.
stability (t50 values) increased from ∼35 to ∼59 °C in the The medium-chain ADHs are homologous, and we identified
enzyme with the G229S, L232V, D236N, and V242I the common core elements among 13 three-dimensional
substitutions and additionally increased to ∼72 °C with the structures of diverse dimeric and tetrameric ADHs (Table 2),
M168R and V173A substitutions. Site-specific substitutions some with <25% sequence identity (Table 3S of the Supporting
should be used to determine which of these residues are Information). The common core has 248 amino acid residues
responsible. Introduction of several additional electrostatic in structurally similar elements (Table 4S of the Supporting
interactions between subunits in the mesophilic ADH from Information). Although the structures are similar, the enzymes
Clostridium beijerinckii increased themostability (T1/2) only differ in substrate specificity and catalytic activity.
from 63.8 to 69.5 °C and did not yield the stability found for Zinc Content and Coordination. Each subunit of yeast
the homologous thermophilic ADH from Thermoanaerobacter ADH1 in the crystals contains a “catalytic” zinc and a
brockii of 93.8 °C.30 “structural” zinc, with no known functional role. Determination
Structural Similarities among Medium-Chain ADHs. of the number of zinc atoms in yeast ADH1 has produced
Comparison of dehydrogenase structures provides information differing results (see the Supporting Information), but the
about evolutionary and structure−function relationships.23,31 crystallography clearly shows that two zincs per subunit can be
Progressive sequence alignments showed only ∼24% sequence present. The coordination of these zincs is tetrahedral, and the
identity between yeast and horse ADHs,32 but detailed distances are typical for ADHs; however, the catalytic zincs in
molecular modeling showed that the three-dimensional subunits A and C ligate TFE, with Glu-67 OE2 in the second
structures were probably very similar, with conservation of sphere, and the zincs in subunits B and D ligate Glu-67 OE2,
space filling in hydrophobic cores, glycine residues, zinc ligands, with TFE in the second sphere (Table 5S of the Supporting
and catalytic residues in the active site.11 Nevertheless, 14 Information).
deletions (or insertions) in various loops, including the With a closed conformation, one subunit (A or C) in each
apparent deletion of 21 amino acids in yeast ADH asymmetric dimer binds NAD and TFE with the catalytic zincs
corresponding to residues 119−139 in the liver enzyme, coordinated in the “classical” manner with Cys-43, His-66, Cys-
indicated that the structures were different in many places. 153, and the oxygen of TFE (Figure 2A). This figure also shows
Comparison of the actual three-dimensional structures (PDB that the methylene carbon of TFE would have its pro-R
entries 4DXH and 4W6Z, A chains) shows that 312 residues of hydrogen directed toward the re-face of the nicotinamide ring.
horse and yeast ADHs are in similar secondary structures and In an open conformation, the other subunit (B or D)
that 11 of the 15 changes we identified agree in most respects coordinates the catalytic zinc in an “alternative” manner, to
with the modeling (Table 1). Good models can be useful Cys-43, His-66, Glu-67, and Cys-153 with the oxygen of TFE in
approximations for homologous structures, but experimental the second sphere (Figure 2B). An overlay of the catalytic
verification is needed. An automated multiple-sequence align- domains of subunits A and B shows that the zinc coordination
ment located 9 of the 15 differences at positions similar to is inverted, accompanied by small movements of the zinc
those found by comparison of the three-dimensional ligands, as the zinc moves ∼2.6 Å closer to Glu-67.
structures.32 Almost all of the insertions or deletions are in The classical and alternative coordinations of the catalytic
the loops connecting α-helices and β-sheet strands and thus do zincs are also found in other ADHs. Of particular interest is the
not alter the overall folding of the protein (Table 1). fact that subunits of human ADH3 complexed with NADH
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Biochemistry Article

Table 2. Common Structural Elements in Horse and Yeast clinic form) also has an asymmetric dimer, and nucleotide is
ADHs Interrupted by Insertions or Deletionsa bound to only one subunit.36 If the asymmetry in the crystal of
yeast ADH reflects a preexisting state that is present in solution,
similarities differences
biochemical studies could provide information about the
location of stoichiometry or cooperativity of coenzyme binding.
horse yeast horse yeast change
Accurate determination of the number of binding sites for
1−6 1−3 amino terminal coenzyme is uncertain because it depends upon having “pure”
7−57 4−54 58 55−57 substrate enzyme, probably with the full complement of eight zincs per
binding site
59−94 58−93 95 94, 95 start of
tetramer and sulfhydryl groups in the proper state of oxidation,
structural Zn and knowing the enzyme concentration and the molecular
loop weight of the tetramer (see the Supporting Information).
96−113 96−113 114−143 114−122 excursionary Various studies have given results that range from 2.0 to 3.6
loop
NADH molecules binding per tetramer. In numerous
144−183 123−162 184, 185 163 connect αA, βA
preparations of recombinant yeast ADH1 in our laboratory,
186−199 164−177 200 178, 179 connect βA, αB
we used titration with NAD+ in the presence of 10 or 100 mM
201−216 180−195 217, 218 196 connect αB, βB
pyrazole to determine the concentration of active sites for
219−247 197−225 248, 249 226 connect βC,
αCD calculation of turnover numbers. The NAD+−pyrazole complex
250−283 227−260 284, 285 261 connect αE, βE forms tightly (Kd ∼ 1 μM, too tight to obtain a good value for
286−296 262−272 297, 298 273 connect βE, βS Kd with micromolar enzyme concentrations) and absorbs light
299−309 274−284 310 285, 286 connect βS, βF with maxima in the difference spectrum at 285 and 293 nm,
311−319 287−295 320−323 296 connect βF, α3 with a difference extinction coefficient at 293 nm of ∼12000
324−339 297−312 340−347 313−317 connect α3, M−1 cm−1, so that the concentration of active sites is readily
βI:5 estimated for solutions of ∼1 mg/mL enzyme in split cuvettes
348−366 318−336 367 337−338 connect α4, with a path length of 0.435 cm.41−43 (A structure of the horse
βI:6 liver enzyme complexed with NAD+ and pyrazole shows a
368−374 339−345 346, 347 carboxyl partial covalent bond between C4N of the nicotinamide ring
terminal
a and a N of pyrazole, with the other N of pyrazole ligated to the
The structures of the ternary complexes of yeast (PDB entry 4W6Z)
catalytic zinc; the difference absorbance maximum is relatively
and horse liver (PDB entry 4DXH) with NAD and 2,2,2-
trifluoroethanol, A chains, were superimposed with O, using 16 flat at 284−296 nm.44) With many batches of purified yeast
common structural elements with 248 residues (Table 4S of the ADH, we found concentrations of active sites that averaged
Supporting Information). Inspection of the structures identified the 72% (range of 50−90%) of the concentration of protein
similar structural elements (helices, β-strands, and loops), as described subunits calculated from the ε280 of 1.26 cm−1 mg−1 mL. We
previously,2 and the sites of insertions and deletions. Differences of ∼3 suggest that such titrations establish that the enzyme can bind
Å in Cα positions were tolerated, as there are shifts in the structural one coenzyme per subunit, but heterogeneity in the enzyme
elements, with retention of the conformation. PDBeFold on the preparations can decrease the observed capacity. Such
EMBL-EBI server aligned 319 residues in 21 structural elements with titrations, however, do not eliminate the possibility that binding
an rmsd of 2.3 Å, but the structure differed at 11 positions because of of coenzyme is cooperative. Numerous studies show that
shifts of a residue in the alignment.
enzyme activity with ethanol and acetaldehyde also fits
Michaelis−Menten kinetics, but such studies do not define
(PDB entries 1TEH and 1MC5) or ADP-ribose (PDB entry the number of sites that are active at any moment. Radiation
2FZE) apparently have both the classical and alternative inactivation studies suggest that each monomer is active.45
coordinations, with crystallographic evidence for partial Because yeast ADH1 appears to be able to bind one NAD
occupancy at two different positions for the zinc separated by per subunit, can the asymmetry in the crystal structure be
2.3 Å.37 At low resolution, it can appear that the zinc has a explained by the preparation of the crystals? It is relevant that
bipyramidal coordination.38,39 The structures with human the crystals were soaked for several days and shipped in a
ADH3 can illustrate an intermediate in the mechanism after mother liquor that contained no added NAD+. The dissociation
the coenzyme is bound and before the substrate (e.g., alcohol) constant for NAD+ is 920 μM, and the Ki for TFE is 2.8 mM;
binds.40 It is clear that the alternative coordination is common therefore, most (96% by estimation for an ordered mechanism)
and that the active site zinc coordination is flexible, which leads of the enzyme should be in the ternary complex in solution
to a proposal for a mechanism by which zinc-bound water is during the crystallization.8 However, subsequent soaking of the
replaced by an alcohol or aldehyde (see below). crystal in buffer without coenzyme could allow most of the
Asymmetry within the Tetrameric Molecule. The coenzyme to dissociate. Nevertheless, if crystal lattice contacts
asymmetry within the dimeric units of yeast ADH1 raises maintained the subunit in the closed conformation, coenzyme
questions about potential cooperativity in the catalytic dissociation should be prevented (as observed in subunits A
mechanism of the molecule, in particular, “half-of-the-sites” and C), and if the crystal lattice contacts held one subunit in
reactivity. However, the dimers are the crystallographic the open conformation, the affinity for coenzyme could be
asymmetric units, and the subunits can be different because greatly diminished. For instance, with horse liver alcohol
of the crystal lattice contacts. The horse liver ADH1E dehydrogenase, mutations that seem to “lock” the subunits
holoenzyme3 and the cod liver ADH125 also have a dimer as “open” increase the Kd for NAD+ from 27 to 1100 μM (and the
the asymmetric unit, and both subunits bind coenzyme; Kd for NADH from 0.50 to 320 μM), and the position of the
however, the subunits in the cod enzyme have different extents nicotinamide riboside is not clearly defined in the structure.46
of closure of the cleft between the coenzyme and catalytic Examination of the crystal lattice contacts for yeast ADH1
domains. The yeast cinnamyl alcohol dehydrogenase (mono- suggests that the contacts around subunits A and C are
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Biochemistry Article

Figure 2. Stereoviews of the two different types of coordination of the catalytic zinc. (A) Classical found in subunits A and C, which has TFE ligated
to the zinc, bound NAD, and Glu-67 5 Å from the zinc (gray sphere). (B) “Alternative”, found in subunits B and D, in which TFE interacts with His-
66 and not with the zinc, but Glu-67 is ligated to the zinc, and no NAD is bound. The electron density maps are contoured at 1.5σ. These figures
were made with the PyMOL Molecular Graphics System, version 1.7.

Figure 3. Active site in subunit A showing the proton relay system, including the alcohol ligated to the catalytic zinc. His-48 forms a hydrogen bond
with nicotinamide ribose O3′ but can readily swing to form a hydrogen bond with ribose O2′ and complete the proton relay system from the bound
alcohol and His-48. This figure was made with the Molray web interface in Uppsala, Sweden.117

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Biochemistry Article

somewhat different than those around the B and D subunits, with Gly-181 N, O2A with His-44 ND1, O1N with Leu-182 N,
consistent with the asymmetry, and it appears that coenzyme and O2N with Arg-340 NH1. O1A also interacts with Gly-339
could dissociate from the open B and D subunits. Although the O and Arg-340 NH1 via a water. O1N also interacts with a
asymmetry in the structure of yeast ADH1 could result from water that interacts with Ala-245 O, Gly-177 O, and Gly-183 N.
inherent structural differences in solution, there is no evidence These interactions are similar to those found for the horse liver
of “negative” cooperativity in the binding of coenzymes or the structures.52,64 Water molecules in the coenzyme binding
kinetics of catalysis.8,47−50 It seems most likely that the enzyme interface are also found in many dinucleotide binding domains
crystallized in the asymmetric form and that coenzyme and may be important for specificity and modulating affinity.65
dissociated from the subunits in the open conformation when The H44R substitution in yeast ADH1 decreased the
the crystals were soaked without coenzyme. In any case, we dissociation constants for NAD+ and NADH by 2−4-fold and
believe that the different structures of the subunits represent decreased turnover numbers by 4−6-fold.18 Although these
energetically accessible states that fortuitously provide signifi- effects are small, they are physiologically significant because the
cant information for the catalytic mechanism. The differences decreased rate of reduction of acetaldehyde apparently leads to
in energy between the open and closed apoenzyme or a more reduced intracellular state during fermentation and
enzyme−NAD+ complexes of horse liver ADH are calculated protects the yeast against toxicity due to oxidation of allyl
to be relatively small,28,51 and this may be true for yeast ADH. alcohol to acrolein.66 Aerobic growth of yeast in the presence of
Active Site in Subunits A and C. Coenzyme and the allyl alcohol is inversely correlated with the catalytic efficiency
substrate analogue, TFE, bind to one of the two subunits in of six different ADH mutants, whereas anaerobic growth in the
each dimer, in what appears to be a mimic of a Michaelis absence of allyl alcohol is positively correlated with the catalytic
complex (Figure 3). The structure is very similar to that found efficiency.
in the holoenzyme complex of horse liver ADH (PDB entry As compared to horse liver ADH1E, yeast ADH1 has 40−
4DXH).52 The TFE alcohol oxygen is ligated to the zinc, and 100-fold faster turnover and 30−60-fold weaker binding of
the methylene carbon is ∼3.7 Å from C4N of the coenzyme coenzymes, and it is of interest to identify the responsible
ring with the pro-R hydrogen directed toward the re face of the structural features (in addition to His-44). Enzymes binding
nicotinamide ring as expected for the known stereochemistry.53 NAD or FAD have the signature sequence GXGXXG within
(The distance was determined at 1.12 Å resolution in the horse the ADP binding βαβ-fold that connects the first β-strand to
liver enzyme to be 3.44 ± 0.02 Å.54) The oxygen of TFE is the first α-helix and makes a tight turn to accommodate the
hydrogen-bonded to OG1 of Thr-45, which is in turn coenzyme.67,68 The sequence in horse liver ADH consists of
hydrogen-bonded to O2′ of the nicotinamide ribose. This residues 199−204, GLGGVG, whereas in yeast ADH1 it
constitutes the inner part of the proposed proton relay system consists of residues 177−183, GAAGGLG. Converting the Ala-
that links the buried alcohol to solvent, as discovered in the Ala sequence in yeast ADH1 to Leu with the A178Dele-
horse liver enzyme.52,55 In contrast to the structures of the tion:A179L substitution (AA:L) decreased by 5−23-fold the
horse liver enzyme, where imidazole NE2 of His-51 interacts affinity for NAD+ (Kia) and the turnover number and catalytic
with the O2′ ribose hydroxyl group and could act directly as a efficiency for ethanol oxidation.69 Comparison of the yeast and
base, imidazole NE2 of His-48 in yeast ADH interacts with the horse ADH structures suggests that the AA:L substitution
O3′ hydroxyl group of the nicotinamide ribose, while ND1 should have been readily accommodated, which perhaps fits
interacts with OD1 of Asp-53. This conformation puts the with the small changes in kinetics. The GXGXXG motif
imidazole CD2 closest to the O2′ ribose hydroxyl group, but sometimes has Ala instead of the final Gly. In yeast ADH1, the
flipping the imidazole group would place ND1 in a hydrogen G183A substitution decreased the affinity for coenzymes and
bond with the O2′ hydroxyl and allow His-48 to act as a base. adenosine nucleotides, and turnover numbers and catalytic
Alternatively, unhindered rotation about the χ1 angle of His-48 efficiencies by 10−400-fold.69 Addition of the methyl group
could place NE2 in a hydrogen bond with the O2′ hydroxyl, in (with G183A) would create steric hindrance with Gly-177 and
position to act as a base, at 2.6 Å. Ala-178 that would distort the structure and affect activity.
Modification of approximately one histidine per subunit with At the end of the second β-strand of the βαβ-fold in NAD-
diethyl pyrocarbonate inactivates the enzyme, with a rate dependent ADHs is usually a conserved aspartic acid residue,
constant increasing with pH and a pK value of 7.1, apparently Asp-201 in yeast ADH (Asp-223 in horse), which forms
because a histidine is involved in ternary complex formation hydrogen bonds with O2′ and O3′ of the adenosine ribose and
rather than binding of coenzyme.56−58 The H48Q substitution interferes with binding of NADP. Lys-206 (Lys-228 in horse
decreases the catalytic efficiency for oxidation of ethanol by 11- ADH) also interacts with O3′ of the adenosine ribose. The
fold and alters the pH dependence.59,60 The change in activity critical role of Asp-201 was demonstrated with the D201G
is consistent with a role for His-48 in catalysis, similar to that substitution, which eliminates the specificity for NAD+, and
found for the horse liver enzyme with the H51Q substitution, allows the enzyme to use NADP+ about as well as NAD+;
which affects both coenzyme binding and subsequent catalytic however, the affinity for NAD+ decreases by ∼15-fold, and the
reactions.61,62 turnover number for ethanol oxidation decreases by ∼8-fold.70
The two cysteines at the active site that chelate the catalytic The conserved aspartate is replaced with a neutral residue (Gly,
zinc, Cys-43 and Cys-153, are chemically modified by different Ala, Val, Ser, Thr, Asn, and Leu), and one of the next two
reagents with loss of activity.7,63 The structure shows that the residues is usually an Arg in NADP-dependent ADHs. In an
modifications would disrupt binding of substrates and affect attempt to improve the catalytic activity of yeast ADH with
ternary complex formation with coenzymes and substrate NADP, the G203R and D201G:G203R enzymes were created.
analogues. The G203R enzyme had kinetic constants quite similar to those
Coenzyme Binding. The coenzyme is bound in the of the wild-type enzyme, whereas the kinetic constants for the
extended conformation typical of ADHs. The pyrophosphate D201G:G203R enzyme with NAD+ or NADP+ were very
oxygen atoms interact with several NH groups: O1A interacts similar to those for the D201G enzyme.69 Apparently, the
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Biochemistry Article

Figure 4. Substrate binding pocket, in subunits A and C. This figure was made with the Molray web interface in Uppsala, Sweden.117

introduced Arg-203 is exposed to solvent and does not swing structures may more successfully explain the differences in
into place to interact with the 2′-phosphate of NADP. activity of the yeast and horse ADHs.
The adenine ring of the NAD+ bound to yeast ADH is Substrate Binding Site. The substrate binding site is
sandwiched between Phe-221 on one side and Ser-246, Ser- illustrated in Figure 4. Several large hydrophobic residues, Trp-
248, and Ala-251 on the other side, as compared to the more 54, Trp-92, Met-270, and Tyr-294, produce a cavity that
hydrophobic residues in horse liver ADH (Phe-198, Ile-224, accommodates ethanol as the best substrate.6 Longer,
and Ile-269). [NAD and 8-I-NAD should bind similarly, as the branched, or secondary alcohols are poorer substrates for
8-iodo substituent of 8-I-NAD is exposed to solvent, but close ADH1.72,73 The catalytic efficiencies (V/Km substrate) of the
(4.1−4.2 Å) to CB of Val-247.] Attempting to increase the three recombinant ADHs from S. cerevisiae on primary alcohols
affinity of yeast ADH for coenzymes, we introduced the S176F, decrease with chain length, but the M270L substitution in
G202I, and S246I substitutions, which could have increased the ADH1 (yeast ADH numbering, Val-294 in horse ADH)
hydrophobic character near the adenine ring, but for all three increases activity on pentanol and hexanol by 10-fold, without
enzymes, the affinity for coenzymes decreased!69 The kinetics affecting activity on ethanol or propanol, and reproduces the
of the G202I enzyme were similar to those of wild-type ADH1; pattern of specificity found in ADH2 and ADH3, which have
however, turnover numbers for the S176F enzyme decreased Leu-270.8 However, ADH2 has 8-fold higher activity than
∼10-fold, and turnover numbers for the S246I enzyme ADH1 on ethanol, which must be due to substitutions of
decreased ∼350-fold. Inspection of the yeast ADH1 structure residues that are not in the active site and probably affect
suggests that Ile-202 might interact with Phe-221 (in contact dynamics of catalysis. Early mutagenesis studies showed
with the adenine ring) but not generate new interactions with surprisingly modest effects (∼5-fold) of T45S, W92F, W92A,
T45S:W92F, and T45S:W92A substitutions on specificities of
the adenine ring. Phe-176 would apparently fit into the adenine
oxidation of the series of primary alcohols from ethanol to
binding pocket if Tyr-258 could swing out of its position, so
hexanol.74 Subsequent studies showed that the single T45S and
there might be some small structural rearrangements that
W54M substitutions and the triple T45S:W54M:W92A (to
account for the relatively large changes in kinetics. The S246I
mimic human75 and monkey ADH1A, αα-isoenzyme) sub-
change is the most enigmatic because it would seem that Ile- stitution decrease activity on primary alcohols relative to that of
246 could interact well with the adenine ring, but the S246I wild-type ADH1, and activity decreases as chain length
enzyme exhibited very large decreases in catalytic efficiency. For increases.72 In contrast, the W92A and T45S:W92A sub-
comparison, I224G (Gly-202 in yeast) and I269S (Ser-246 in stitutions decrease activity on ethanol by ∼400-fold but
yeast) substitutions in horse liver ADH decreased the affinity increase activity by 3−9-fold on hexanol (an ∼1000-fold
for both coenzymes 60- and 350-fold, respectively, and inversion of activity), with a pattern of activity that resembles
increased the turnover number for ethanol by 7- and 26-fold, that observed with monkey ADH1A.72 The W92A substitution
respectively, but did not change the catalytic efficiency for also confers weak activity on cyclohexanol (no detectable
ethanol oxidation or acetaldehyde reduction.71 The increases in activity with wild-type yeast ADH1), but the activity is less than
turnover numbers are due to faster release of NADH, which is 1/105 of that of monkey ADH1A. Increasing the size of the
rate-limiting for the horse enzyme. It is satisfying that the horse binding pocket with these several substitutions and the W54L
I224G ADH has binding constants for coenzymes that are substitution also increases activity on branched-chain alco-
similar to those for wild-type yeast ADH, but the reverse G202I hols.76
substitution in yeast ADH also has dissociation constants Engineering the substrate specificity of yeast ADH based on a
(lower affinity) larger (5−9-fold) than those of wild-type yeast model constructed from the horse liver enzyme structure gave
ADH.69 Comparing the results for the mutagenesis studies on some patterns of activity that were expected, but changes in
the yeast and liver enzymes suggests that it is easier to diminish catalytic efficiencies are difficult to predict. Comparison of the
catalytic activity than to enhance it. More extensive, and yeast and horse enzyme active sites suggests that this is due in
multiple, substitutions that are designed on the known part to the insertions or deletions (Trp-54 and Tyr-119) near
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Biochemistry Article

the active site and to differences in rotamers (Trp-92), the ∼100-fold and shifted pH dependencies.88 The results from the
peptide backbone, and the particular amino acid residues at and site-directed substitutions are consistent with the proposed
near the active site. mechanism, but electrostatic effects and local, small structural
Yeast ADH1 is highly specific for transfer of the (4R)- changes could also affect the dynamics of catalysis.
nicotinamide hydrogen of NADH to aldehydes and the pro-R Although a structure for the binary yeast ADH1−coenzyme
hydrogen from ethanol or octanol to NAD+.77−79 However, it complex is not available, we propose, by analogy to human
has weak activity (1/800 of that on ethanol) on 2-propanol, ADH3 structures (PDB entries 1TEH and 1MC5), that the
which indicates that the enzyme can accommodate (poorly) a binding of Glu-67 to the zinc may be weakened when the
methyl substituent on the oxidizable carbon.72 ADH1 has coenzyme binds because Glu-67 interacts with Arg-340, which
highly preferential activity on (S)-2-butanol as compared to in turn interacts with a phosphate oxygen of the coenzyme.
(R)-2-butanol, and it appears that the methyl group of (S)-2- Such an interaction is common in most tetrameric ADHs
butanol must be accommodated near Trp-92.72,80 Yeast ADH complexed with NAD(P)(H). In apoenzyme subunits (B and
does not measurably oxidize secondary alcohols with both D) of yeast ADH1, Glu-67 is ligated to the zinc and does not
substituents larger than a methyl group, so that there are limits interact with Arg-340, but in the holoenzyme subunits (A and
to the flexibility of the active site.81 C), Glu-67 interacts with Arg-340. In the proposed binary
Mechanistic Implications of Asymmetry and Alter- enzyme−coenzyme complex, ligand exchange of water, alcohol,
native Coordination of Catalytic Zinc. The two different and aldehyde would be facilitated by a modulated interaction of
conformational states of the subunits can represent two Glu-67 with the zinc.
relevant structures in the mechanism, the apoenzyme and the The binding of alcohol to the zinc to form ternary complexes
ternary complex. The kinetic mechanism for the yeast enzyme (of horse liver ADH) appears to require deprotonation of the
acting on ethanol appears to be ordered (or preferred ordered) zinc-bound water in the enzyme−NAD+ complex before
in which coenzyme binds to free apoenzyme subunits with the ligands bind to the zinc.84 This deprotonation appears to be
open conformation and then isomerizes to form the closed facilitated by a proton relay system (see Figure 3 for yeast
conformation.8,82 The isomerization step has been described ADH) in the hydrogen-bonded system that includes the zinc-
for the horse liver enzyme.83,84 Transient kinetic evidence is bound water, the hydroxyl group of Ser-48, the 2′-hydroxyl
lacking for an enzyme−coenzyme isomerization for the yeast group of the nicotinamide ribose, and the imidazole group of
enzyme, but effects of pressure on V/K for benzyl alcohol His-51.55,62 Dissociation of hydroxide from zinc is likely to be
oxidation led to the suggestion that the E−NAD+ ↔ E*− less favorable than release of water, but displacement by the
NAD+ equilibrium constant is 75 ± 13.85 Hydrogen−deuterium glutamate carboxylate could be energetically favorable. After a
exchange studies also suggest that binding of NAD+ results in a neutral alcohol binds to the zinc and displaces the glutamate,
conformational change.86 the alcohol would be deprotonated via the proton relay system
The next step in the mechanism, the binding of alcohol to to form the zinc alkoxide that is the ground state for the
the enzyme−NAD+ complex, may occur by direct displacement hydride transfer step.28,46,83 The dissociation of aldehyde and
of Glu-67 by the alcohol or by a double displacement of a water binding of water could also occur by an inversion of the zinc
that could bind to the zinc in the enzyme−NAD+ complex. The coordination, which is illustrated by the complexes of human
homologous E. coli enzyme complexed with NAD has water ADH3 with NADH.
bound to the catalytic zinc.26 The mechanism for ligand An alternative mechanism for ligand exchange could involve
exchange in ADHs is controversial. The observation of the a transient pentacoordinated zinc.89 Pentacoordinate zinc was
alternative zinc coordination (Figure 2B) and the many observed in the binary complex of horse liver ADH with 1,10-
examples of such coordination in other ADHs (Table 6S of phenanthroline, indicating some flexibility of the zinc
the Supporting Information) are significant because they coordination.90 However, the first structures of ternary
indicate that the zinc coordination can change during catalysis complexes (coenzyme and substrate analogue) showed that
and can lead to a reasonable mechanism for the exchange of no water remained bound to the zinc.3,55 Nevertheless, such a
ligands. Computational studies suggest that the carboxyl group pentacoordinated zinc could be a transient species during the
of Glu-68 in liver ADH (Glu-67 in yeast ADH) could replacement of the water with the alcohol. A pentacoordinated
transiently displace a zinc-bound water and then allow the zinc has not been observed in any ternary complexes of ADHs,
alcohol to bind by displacing the carboxyl group.87 This ligand except for the complex of sorbitol dehydrogenase with a
exchange mechanism is more energetically feasible than chelating (“transition state analogue”) inhibitor that mimics the
associative (tight, intermediate five coordination) or dissocia- substrate sorbitol (PDB entry 1P16).91
tive (intermediate three coordination) mechanisms. The Another version of a mechanism involving a pentacoordinate
exchange would occur as the zinc moved closer to Glu-67, to zinc was proposed to account for alternative waters bound to
form a loose, transient intermediate, trigonal−bipyramidal the catalytic zinc and to C6N of the nicotinamide ring in binary
pentacoordinate zinc with the water and glutamate in axial complexes with NADH, but this mechanism is not supported
positions.40 The configuration of the zinc is essentially inverted, by structures of ternary complexes.92,93
and then alcohol displaces the Glu to restore the configuration. Time-resolved X-ray absorption studies of the transient
The alternative zinc locations in human ADH3 structures reaction of T. brockii ADH with NADP+ and the substrate 2-
provide evidence of an exchange mechanism.37−40 Moreover, propanol showed that two different pentacoordinate species
the substitution of the highly conserved Glu32 with Leu in formed at 5 and 15 ms (during the “burst” phase) but then
ADH3 decreased catalytic efficiency by 3000-fold for oxidation perhaps reverted (to tetracoordination) during the steady state
of (S)-hydroxymethylglutathione, without large changes in the phase.94 These results could fit with the X-ray structure of the
protein structure or binding of the coenzyme.37 Likewise, for complex with NADPH, where the zinc is essentially
yeast ADH1, the E67Q substitution decreased the catalytic bipyramidal with the oxygens of Ser-39 and Glu-60 ∼3.7 Å
efficiency for ethanol oxidation or acetaldehyde reduction by from the zinc, and 2-propanol displaces the Ser oxygen. A
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Biochemistry Article

mechanism that involves displacement of Glu-60 with water Accession Codes

and then binding of alcohol to form the reactive complex was The X-ray coordinates and structure factors have been
proposed, but the water was not assigned a catalytic role. The deposited in the Protein Data Bank as entry 4W6Z (making
proposed mechanism seems to be more elaborate than the earlier deposition of 2HCY obsolete). Coordinates for the
chemically necessary. Nevertheless, evidence of transient biological tetramers can be generated by applying the rotation
species is required to describe the exchange mechanism. matrix found in the header of the PDB file.
Experimental support for a catalytic role of Glu-60 in this
enzyme is limited as the substitution with Ala or Asp decreases
the catalytic efficiency by only 4- or 8-fold, respectively.95 The
■ AUTHOR INFORMATION
Corresponding Author
mechanisms for ligand exchange may differ in the enzymes with *E-mail: [email protected]. Phone: (319) 335-7909. Fax:
two carboxylates bound to the zinc instead of two cysteine (319) 335-9570.
sulfhydryl groups.
Present Addresses
Transition State Studies. The transition state of yeast †
ADH has been extensively characterized. Structure−activity S.B.R.: Lawson State Community College, Birmingham, AL
35216.
correlations for para-substituted benzyl alcohols and benzalde- ‡
S.R.: Institute for Stem Cell Biology and Regenerative
hydes suggested a transition state with little change in charge
Medicine (inSTEM), National Center for Biological Sciences,
relative to that of the alcohol, whereas α-secondary isotope
GKVK Post, Bellary Road, Bangalore 560065, India.
effects suggested a transition state that resembled alde-
hyde.96−99 Other studies provided values for intrinsic isotope Funding
effects.100−103 In a landmark study, yeast ADH was used to This work was supported by NSF grant MCB 9118657 and
show that the transfer of hydrogen from benzyl alcohol to NIH grant AA00279.
NAD+ occurs with quantum mechanical tunneling.104 Sub- Notes
sequent studies with horse liver and bacterial ADHs show that The authors declare no competing financial interests.
the tunneling, along with coupled motions, is a general
phenomenon that can explain the results with yeast
ADH.105−109 Pressure diminishes substrate and solvent isotope
■ ACKNOWLEDGMENTS
We thank Kristine B. Berst for protein chemistry and The
effects, perhaps by affecting a “mechanical” component University of Iowa Molecular Analysis Facility for amino acid
involving vibrational dynamics.110−113 These early studies and molecular mass analyses. We also appreciated the
used benzyl alcohol as a substrate (where hydrogen transfer numerous efforts by Darla Ann Kratzer, Susan Souhrada,
is rate-limiting for catalytic turnover because of small Andrew D. Hershey, Fan Fan, Doo-Hong Park, Suresh Pal,
“commitment factors”) and commercial yeast ADH, which Henry A. Charlier, Jr., and Mark Hermes to prepare crystals of
contains mostly ADH1, but which has ∼40000-fold more yeast ADH. We thank Lokesh Gakhar in The University of
activity on ethanol than on benzyl alcohol.73 In contrast, yeast Iowa Crystallography Facility for advice. We also thank the staff
ADH2 is ∼100-fold more active than ADH1 on benzyl alcohol; and support at beamline BW7A at the EMBL/DESY Hamburg
fortunately, the structure−activity relationships for ADH1 and synchrotron. S.R. collected the data while working in the
ADH2 with the substituted benzyl alcohols and benzaldehydes Department of Molecular Biology at the Swedish University of
are similar.73 Subsequently, the secondary and equilibrium Agricultural Sciences (Uppsala, Sweden) and determined the
isotope effects for reduction of substituted benzaldehydes were structure at The University of Iowa.

■
determined for yeast ADH2, and a model that describes the
“tunneling ready state”, explains the isotope and substituent ABBREVIATIONS
effects, and fits the three-dimensional structure was created.114 ADH, alcohol dehydrogenase; TFE, 2,2,2-trifluoroethanol
Interestingly, the distance between the methylene carbon of the (ETF in the PDB entry, with the methylene carbon labeled
alcohol and C4N of the nicotinamide ring was calculated to be “2”); rmsd, root-mean-square deviation; amino acid substitu-
3.2 Å, which is close to the distance of 3.8 Å for the structure tions, e.g., D236N, Asp-236 substituted with Asn.

■
with TFE. Benzyl alcohol can fit with some close contacts into
the active site, in particular with the M270L substitution,73 and REFERENCES
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